1. Field of the Invention
The present application relates to the field of biotechnology and, in particular, to the fields of cloning and protein expression.
2. Related Art
The fundamental process that sustains the ongoing biotechnology revolution is the cloning of DNA molecules for their further analysis or use. Cloning of DNA molecules has been practiced in the art for many years. A typical cloning protocol will involve identifying a desired DNA molecule, preparing a population of recombinant vectors by ligating the DNA molecule with a vector in a mixture of DNA molecule, vector and an appropriate ligase enzyme, transforming the population of recombinant vectors into a competent microorganism, growing the microorganism for some period of time sufficient to permit the formation of colonies, selecting colonies of microorganisms that potentially contain the desired DNA molecule correctly ligated in the vector, growing a sufficient quantity of each selected colony from which to isolate the recombinant vector, analyzing the isolated vector to ensure that the vector contains the desired DNA molecule and then growing a sufficient quantity of the microorganism that contains the correct recombinant vector to perform whatever subsequent manipulations are required. For details of various cloning procedures the reader may consult Sambrook, et al. 1989, Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., specifically incorporated herein by reference
The typical cloning protocol outlined above thus includes at least three steps that involve growing of a microorganism. Since these growing steps generally require 12-16 hours and are usually performed as overnight incubations, the rate limiting steps for experiments involving cloning of a DNA fragment are the steps requiring growth of a microorganism. Although there are many variations on the basic practice of cloning, virtually all cloning methods require the insertion of the DNA molecule of interest into a microorganism and growth of the microorganism and, therefore, the speed of virtually every cloning methodology is limited by the rate of growth of the microorganism used for cloning.
For most cloning applications, the microorganism of choice is Escherichia coli (E. coli. Although numerous strains of E. coli are known, most cloning applications use one or another derivative of E. coli K-12. These derivatives suffer from the slow growth rate discussed above. Other known strains of E. coli, such as E. coli W (ATCC9637), have a rapid growth rate when compared to E. coli K-12; however, wild type strains of E. coli W and other rapid growing strains are not suitable for biotechnology applications for several reasons. First, the genetics of the organism have not been determined to the level of detail required by cloning applications. Thus, those skilled in the art would not know whether the genome of a microorganism contained the appropriate modifications of a number of genes that would make the microorganism suitable for biotechnology applications. For example, microorganisms are generally recA+ which leads to the formation plasmid multimers and makes the microorganism less suitable for applications that involve the isolation of plasmid. Microorganisms typically contain numerous protease genes and may degrade overexpressed proteins thereby decreasing the yield of a desired protein product. Microorganisms typically contain a lac operon that does not permit alpha complementation and, therefore, the identification of recombinant vectors is more difficult. Further, many microorganisms contain endogenous plasmids that complicate the plasmid isolation steps necessary for cloning applications. Finally, microorganisms might contain genes coding for nucleases that could cause the degradation of exogenous plasmids.
For a large number of biotechnology applications, a crucial step in the development of the application involves cloning one or more fragments of DNA. Given the central role of cloning in the development of the biotechnology industry, there has long existed in the art a need for reagents that speed the process of cloning. In particular, there exists a need in the art for microorganisms that have a desirable genotype and a rapid growth rate and can be employed to speed the cloning process. The present invention meets this long felt need.
The present invention provides microorganisms for biotechnology applications characterized by a rapid growth rate as compared to the microorganisms currently used for these applications. In particular, the present invention provides a rapid growing microorganism that preferably lacks endogenous plasmids and is, therefore, suitable for cloning applications. Because the microorganisms of the present invention form colonies faster than the microorganisms currently in use in cloning applications, the present invention provides an improvement in cloning desired nucleic acid molecules, allowing more rapid identification and isolation of recombinant vectors and clones of interest.
The present invention thus provides a method of cloning that employs a rapid growing microorganism. The method entails constructing a population of recombinant vectors, transforming competent microorganisms capable of rapid growth with the population of recombinant vectors, selecting a transformed microorganism containing one or more recombinant vectors of interest and/or isolating one or more recombinant vectors of interest from the transformed microorganism. In one embodiment, the rapid growing microorganism is of the genus Escherichia. In another embodiment, the rapid growing microorganism is an E. coli. In a further embodiment, the rapid growing microorganism is an E. coli strain W. In a preferred embodiment, the rapid growing microorganism is an E. coli strain W lacking endogenous plasmids. In other preferred embodiments, the rapid growing microorganism is selected from a group consisting of BRL3781, BRL3784 and recAxe2x88x92 derivatives thereof. The cloning methods of the present invention may optionally include a step of growing transformed microorganism at an elevated temperature to increase the growth rate of the microorganism, for example, at a temperature greater than 37xc2x0 C. In a preferred embodiment, the transformed microorganisms may be grown at about 42xc2x0 C.
The present invention provides a method of producing a protein or peptide which comprises constructing a recombinant vector containing a gene encoding a protein or peptide of interest, transforming the vector into a competent microorganism capable of rapid growth and culturing the transformed microorganism under conditions that cause the transformed microorganism to produce said peptide or protein. In a preferred embodiment, the rapid growing microorganism is of the genus Escherichia. In another preferred embodiment, the rapid growing microorganism is an E. coli. In another preferred embodiment, the rapid growing microorganism is an E. coli strain W. Other embodiments include a microorganism deleted in the lon protease. In some preferred embodiments, the microorganism carries a gene encoding a 17 RNA polymerase (RNAP). In other preferred embodiments, the T7 RNAP gene is under the control of a salt inducible promoter. In another preferred embodiment, the rapid growing microorganism does not contain endogenous plasmids.
The present invention also includes a method of producing a microorganism for cloning comprising the steps of obtaining a rapid growing microorganism containing endogenous plasmids and curing the microorganism of endogenous plasmids. In a preferred embodiment, the rapid growing microorganism is of the genus Escherichia. In another preferred embodiment, the rapid growing microorganism is an E. coli. In another preferred embodiment, the rapid growing microorganism is an E. coli strain W. In a related aspect of the present invention, any desired modification or mutation may be made in the microorganisms of the present invention including, but not limited to, alteration of the genotype of the microorganism to a recAxe2x88x92 genotype such as recA1/recA13 or recA deletions, a lacZxe2x88x92 genotype that allows alpha complementation such as lacX74 lacZxcex94M15 or other lacZ deletion, a protease deficient genotype such as xcex94lon and/or ompTxe2x88x92, an endonuclease minus genotype such as endA1, a genotype suitable for M13 phage infection by including the Fxe2x80x2 episome, a restriction negative, modification positive genotype such as hsdR17(rKxe2x88x92, mKxe2x88x92), a restriction negative, modification negative genotype such as hsdS20(rBxe2x88x92, mBxe2x88x92), a methylase deficient genotype such as mcrA and/or mcrB and/or mrr, a genotype suitable for taking up large plasmids such as deoR, a genotype containing suppressor mutations such as supE and/or supF. Other suitable modifications are known to those skilled in the art and such modifications are considered to be within the scope of the present invention.
The present invention provides a method of transforming a competent microorganism capable of rapid growth including the steps of obtaining a recombinant vector and contacting a competent microorganism of the present invention with the recombinant vector under conditions which cause the rapid growing microorganism to be take up the recombinant vector. In a preferred embodiment, the rapid growing microorganism is of the genus Escherichia. In another preferred embodiment, the rapid growing microorganism is an E. coli. In another preferred embodiment, the rapid growing microorganism is an E. coli strain W. In another preferred embodiment, the rapid growing microorganism is an E. coli strain W lacking endogenous vectors. The methods of the present invention may optionally include the step of growing the transformed microorganism at elevated temperatures to increase the growth rate of the microorganism, for example, at a temperature greater than 37xc2x0 C. In a preferred embodiment, the transformed microorganisms may be grown at about 42xc2x0 C.
The present invention also includes kits comprising a carrier or receptacle being compartmentalized to receive and hold therein at least one container, wherein the container contains rapid growing microorganisms. The kit optionally further comprises vectors suitable for cloning. In a preferred embodiment, the kits may contain a vector suitable for recombinational cloning. In a preferred embodiment, the rapid growing microorganisms may be competent. In some preferred embodiments, the rapid growing microorganisms may be chemically competent. In other preferred embodiments, the rapid growing microorganisms may be electrocompetent. In some preferred embodiments, the kits of the present invention may include enzyme including, but not limited to, restriction enzymes, ligases, and/or polymerases. In other preferred embodiments, the kits of the present invention may include recombination proteins for recombinational cloning. The kits of the present invention may also comprise instructions or protocols for carrying out the methods of the present invention.
The present invention includes compositions comprising rapid growing microorganisms. In a preferred embodiment, the rapid growing microorganism may be a competent microorganism. In some preferred embodiments, the rapid growing microorganisms may be chemically competent. In other preferred embodiments, the rapid growing microorganisms may be electrocompetent. The compositions of the present invention may optionally comprise at least one component selected from buffers or buffering salts, one or more DNA fragments, one or more vectors, one or more recombinant vectors, one or more recombination proteins and one or more ligases. In a preferred embodiment, the compositions of the present invention may comprise a rapid growing microorganism in a glycerol solution. In other preferred embodiments, compositions of the present invention may comprise rapid growing microorganisms in a buffer. In preferred embodiments, the microorganisms of the present invention may be in a competence buffer. In other preferred embodiments, the compositions of the present invention may comprise a lyophilized rapid growing microorganism.
The present invention includes a method of making competent rapid growing microorganisms comprising the steps of obtaining a rapid growing microorganism, growing the rapid growing microorganism and treating the rapid growing microorganism to make it competent. In some embodiments of the present invention, treating the microorganisms may include the step of contacting the microorganisms with a solution containing calcium chloride. In other embodiments, treating may include the step of contacting the microorganisms with water. Embodiments of the invention may include the step of curing the rapid growing microorganism of endogenous plasmids. In a preferred embodiment, the rapid growing microorganism is of the genus Escherichia. In another preferred embodiment, the rapid growing microorganism is an E. coli. In another preferred embodiment, the rapid growing microorganism is an E. coli strain W.